Cellular test systems for the determination of the biological activities of neurotoxin polypeptides

ABSTRACT

The present invention pertains to a method for the generation of neurotoxin-sensitive, neuronal differentiated cells comprising the steps of: a) cultivating tumor cells which are able to differentiate into neuronal cells in a culture medium under conditions and for a time which primes said tumor cells for neuronal differentiation; and b) cultivating the tumor cells primed for neuronal differentiation of a) in a differentiation medium having an osmolality of 100 to 270 mOsm/kg, and comprising (i) B27 supplement and/or (ii) N2 supplement, for at least 3 days, thereby obtaining neurotoxin-sensitive, neuronal differentiated cells. The invention further relates to neurotoxin-sensitive, neuronal differentiated cells obtainable by the method of the invention. In addition, the invention encompasses a method for determining the activity of a neurotoxin polypeptide comprising the steps of: a) contacting the neurotoxin-sensitive, neuronal differentiated cells obtainable by the method of the invention with a neurotoxin polypeptide; b) cultivating the neurotoxin-sensitive, neuronal differentiated cells of step a) for 3 to 74 hours or 72 hours under conditions which allow for the neurotoxin polypeptide to exert its biological activity; and c) determining the activity of the neurotoxin polypeptide in the said cells after cultivation according to step b). Finally, the invention provides for a medium comprising OptiMEM, FBS, B27 supplement, and N2 supplement.

The present invention pertains to a method for the generation ofneurotoxin-sensitive, neuronal differentiated cells comprising the stepsof: a) cultivating tumor cells which are able to differentiate intoneuronal cells in a culture medium under conditions and for a time whichprimes said tumor cells for neuronal differentiation; and b) cultivatingthe tumor cells primed for neuronal differentiation of a) in adifferentiation medium having an osmolality of 100 to 270 mOsm/kg, andcomprising (i) B27 supplement and/or (ii) N2 supplement, for at least 3days, thereby obtaining neurotoxin-sensitive, neuronal differentiatedcells. The invention further relates to neurotoxin-sensitive, neuronaldifferentiated cells obtainable by the method of the invention. Inaddition, the invention encompasses a method for determining theactivity of a neurotoxin polypeptide comprising the steps of: a)contacting the neurotoxin-sensitive, neuronal differentiated cellsobtainable by the method of the invention with a neurotoxin polypeptide;b) cultivating the neurotoxin-sensitive, neuronal differentiated cellsof step a) for 3 to 74 hours or 72 hours under conditions which allowfor the neurotoxin polypeptide to exert its biological activity; and c)determining the activity of the neurotoxin polypeptide in the said cellsafter cultivation according to step b). Finally, the invention providesfor a medium comprising OptiMEM, FBS, B27 supplement, and N2 supplement.

Clostridium botulinum and Clostridium tetani produce highly potentneurotoxins, i.e. botulinum toxins (BoNTs) and tetanus toxin (TeNT),respectively. These Clostridial neurotoxins (CNTs) specifically bind toneuronal cells and disrupt neurotransmitter release. Each toxin issynthesized as an inactive unprocessed approximately 150 kDasingle-chain protein. The posttranslational processing involvesformation of disulfide bridges, and limited proteolysis (nicking) by thebacterial protease(s). Active neurotoxin consists of two chains, anN-terminal light chain of approx. 50 kDa and a heavy chain of approx.100 kDa linked by a disulfide bond. CNTs structurally and functionallyconsist of three domains, i.e. the catalytic light chain, the heavychain encompassing the translocation domain (N-terminal half) and thereceptor binding domain (C-terminal half); see, e.g., Krieglstein 1990,Eur. J. Biochem. 188, 39; Krieglstein 1991, Eur. J. Biochem. 202, 41;Krieglstein 1994, J. Protein Chem. 13, 49. The Botulinum neurotoxins aresynthesized as molecular complexes comprising the 150 kDa neurotoxinprotein and associated non-toxic proteins. The complex sizes differbased on the Clostridial strain and the distinct neurotoxin serotypesranging from 300 kDa, over 500 kDa, and 900 kDa. The non-toxic proteinsin these complexes stabilize the neurotoxin and protect it againstdegradation; see Silberstein 2004, Pain Practice 4, S19-S26.

Clostridium botulinum secretes seven antigenically distinct serotypesdesignated A to G of the botulinum neurotoxin (BoNT). All serotypestogether with the related tetanus neurotoxin (TeNT) secreted byClostridium tetani, are Zn²⁺-endoproteases that block synapticexocytosis by cleaving SNARE proteins; see Couesnon, 2006, Microbiology,152, 759. CNTs cause the flaccid muscular paralysis seen in botulism andtetanus; see Fischer 2007, PNAS 104, 10447.

Despite its toxic effects, botulinum toxin complex has been used as atherapeutic agent in a large number of diseases. Botulinum toxinserotype A was approved for human use in the United States in 1989 forthe treatment of strabism, blepharospasm, and other disorders. It iscommercially available as Botulinum toxin A (BoNT/A) proteinpreparation, for example, under the trade name BOTOX (Allergan, Inc.) orunder the trade name DYSPORT/RELOXIN (Ipsen, Ltd). An improved,complex-free Botulinum toxin A preparation is commercially availableunder the trade name XEOMIN (Merz Pharmaceuticals, GmbH). Fortherapeutic applications, the preparation is injected directly into themuscle to be treated. At physiological pH, the toxin is released fromthe protein complex and the desired pharmacological effect takes place.The effect of Botulinum toxin is only temporary, which is the reason whyrepeated administration of Botulinum toxin may be required to maintain atherapeutic affect.

The Clostridial neurotoxins weaken voluntary muscle strength and areeffective therapy for strabism, focal dystonia, including cervicaldystonia, and benign essential blepharospasm. They have been furthershown to relief hemifacial spasm, and focal spasticity, and moreover, tobe effective in a wide range of other indications, such asgastrointestinal disorders, hyperhidrosis, and cosmetic wrinklecorrection; see Jost 2007, Drugs 67, 669.

During the manufacturing process of Clostridial neurotoxins, thequalitative and quantitative determination of said neurotoxins as wellas the quality control of the biologically active neurotoxinpolypeptides is of particular importance. In addition, governmentalagencies accept only simple, reliable, and validated Botulinum toxinactivity assays. At present the mouse LD₅₀ bioassay, a lethality test,remains the “gold standard” used by pharmaceutical manufacturers toanalyze the potency of their preparations; see Amon et al. (2001), JAMA285, 1059-1070. However, in recent years, considerable effort has beenundertaken to seek for alternative approaches to alleviate the need foranimal testing and all the disadvantages, costs and ethical concernsassociated with this type of animal-based assays. In addition, theregulatory agencies are engaging pharmaceutical companies to apply thethree “Rs” principle to the potency testing of Botulinum neurotoxins:“Reduce, Refine, Replace”; see Straughan, Ahern. Lab. Anim. (2006), 34,305-313. As a consequence, cell-based test systems have been developedin order to provide reasonable alternatives to methods using liveanimals. Yet, only three cellular test systems are available for thedetermination of neurotoxin biological activity thus far which have beenshown to be sufficiently sensitive to neurotoxin polypeptides. Thesecell-based test systems include the use of primary neurons isolated fromrodent embryos which are differentiated in vitro (Pellett et al. (2011),Biochem. Biophys. Res. Commun. 404, 388-392), neuronal differentiatedinduced pluripotent stem cells (Whitemarsh et al. (2012), Toxicol. Sci.126, 426-35), and a subclone of the SiMa cell line (WO 2010/105234 A1).

However, the isolation of primary neurons requires the killing ofanimals and is laborious and time consuming. Further, test systems usingdifferent primary neurons show large variances. Similarly, thegeneration of neuronal differentiated induced pluripotent stem cells isdifficult and time consuming. In addition, storage of such cells is veryproblematic. Assays using tumor cell lines are frequently not sensitiveenough to BoNT. Moreover, complex differentiation protocols are requiredfor said tumor cell lines which result in large variances and/or highfailure rates of assays using said cell lines.

In light of the above, further test systems for the determination ofneurotoxin polypeptide activity acceptable to governmental agencies andproviding for an alternative to animal-based test systems are highlydesirable.

Thus, the technical problem underlying the present invention may be seenas the provision of means and methods complying with the aforementionedneeds. The technical problem is solved by the embodiments characterizedin the claims and herein below.

The present invention relates, in a first aspect, to a method for thegeneration of neurotoxin-sensitive, neuronal differentiated cellscomprising the steps of:

-   -   a) cultivating tumor cells which are able to differentiate into        neuronal cells in a culture medium under conditions and for a        time which primes said tumor cells for neuronal differentiation;        and    -   b) cultivating the tumor cells primed for neuronal        differentiation of a) in a differentiation medium having an        osmolality of 100 to 270 mOsm/kg, and comprising (i) B27        supplement and/or (ii) N2 supplement, for at least 3 days,        thereby obtaining neurotoxin-sensitive, neuronal differentiated        cells.

In this method of the invention, tumor cells which are able todifferentiate into neuronal cells are first grown in a cell culturemedium under conditions and for a time which primes said tumor cells forneuronal differentiation. Thereafter, the tumor cells thus primed forneuronal differentiation are transferred into a differentiation mediumhaving an osmolality of 100 to 270 mOsm/kg. Further, thisdifferentiation medium comprises at least (i) B27 supplement and/or (ii)N2 supplement. Alternatively, the differentiation medium can compriseNS21 supplement, instead of B27 supplement and/or N2 supplement.Cultivation in said differentiation medium is carried out for at least 3days. Thereby, neurotoxin-sensitive, neuronal differentiated cells areobtained. Preferably, the differentiation medium comprises neurobasalmedium.

As a result of this novel differentiation method or protocol of theinvention, neurotoxin-sensitive, neuronal differentiated cells areobtained which exhibit significantly improved sensitivity to neurotoxinpolypeptides, as shown in detail in the following Example.

Clostridial neurotoxins are characterized in that they specificallyinhibit the secretion of neurotransmitters from pre-synaptic nerveendings. The selectivity for peripheral neurons is mediated by therecognition of two different receptors, SV2 and GT1b. The physiologicaleffect of the neurotoxins is based on the cleavage of a protein of theso-called SNARE complex subsequent to the binding of the receptor andthe translocation of the neurotoxin's light chain. The determination ofthe biological activity of BoNT is an important aspect in thecharacterization of said neurotoxin proteins and is required, interalia, by regulatory authorities for the clearance of BoNT-containingproducts. A reliable test for the measurement of the biological activityof BoNT is, therefore, basis for research, development and marketing ofproducts containing BoNT. Furthermore, cell-based test systems shallreplace the thus far predominant animal tests, for ethical reasons. Forestablishing such cell-based test systems, a sufficient high sensitivityof neuronal cells or cell lines towards Botulinum neurotoxins isessential. However, in order to obtain such high sensitivity, laboriousdifferentiation methods of neuronal cell lines are required so far. As aresult, only a few cell-based test systems are available yet. Todetermine the biological activity of Botulinum toxins in pharmaceuticalproducts, the neuronal cells or cell lines shall have the followingproperties: First, the cells should be of human, neuronal origin inorder to resemble the target as close as possible, i.e. the humanpatient. Second, the cell system shall be robust towards excipients inthe final product and, preferably, also towards impurities inintermediate stages of the production process (process controls). Third,the cell-based test system shall exhibit a dynamic measuring range whichallows for the accurate determination of the biological activity of BoNTin a vial (for example, 50 U BoNT/A). Considering technical factors suchas the solubility of excipients, volumes of cell culture media etc., aBoNT concentration of less than 1 pM has to be determined accurately.According to the inventors' best knowledge, only three cell-based testsystems are available so far which show sufficiently high sensitivity toBoNT. These include primary neurons of embryos from rodents, neuronaldifferentiated induced pluripotent stem cells and a subclone of the SiMacell line, as already mentioned elsewhere herein. However, said celllines have been reported to exhibit a sufficiently high sensitivity,only after complex and laborious differentiation protocols, which arefrequently associated with large variances. In contrast, the presentinvention provides for a simple, reliable and robust cell-based testsystem for the measurement of the biological activity of Botulinumneurotoxins (BoNT) which fulfills the abovementioned requirements andwhich has been further improved, in comparison to the cellular testsystems described in the art.

More specifically, SiMa (human neuroblastoma) and P19 (murine embryonalcarcinoma) tumor cells were first cultivated as described in the priorart, then seeded on multi-well plates and primed for neuronaldifferentiation, as described elsewhere herein and in the followingExample. In a subsequent, novel differentiation step, the cell culturemedium was substituted by a differentiation medium comprising a mediumhaving low osmolality, such as an osmolality of 100 to 270 mOsm/kg. Oneexample for such a medium having low osmolality is neurobasal medium.After a period of differentiation (for example, 3 to 7 days) in whichthe medium was replaced by fresh medium, if applicable, neurotoxinpolypeptide was added to the cell culture medium. Furthermore, GT1b wasused as a sensitivity enhancer for BoNT in the method of the invention.For example, 50 μM GT1b was added to the neurobasal medium upon and/orprior to intoxication of the cells with neurotoxin polypeptide. Afterfurther 72 h incubation with the neurotoxin polypeptide, the cells werestopped and the biological activity of the neurotoxin polypeptide wasanalyzed. As a result, a significant increase in the sensitivity of saidcells to BoNT has been found.

It has surprisingly found by the present inventors that the lowosmolality of the differentiation medium is responsible for theincreased sensitivity to BoNT of the neurotoxin-sensitive, neuronaldifferentiated cells produced by the methods of the invention, asdemonstrated in the following Example. This finding was no trivial taskas evidenced from the history of experiments which finally ended up inthe methods of the invention: At the beginning, the inventors used adifferentiation medium described in the art which comprised MEM+N2supplement+B27 supplement; see differentiation medium 2, in the Example.The sensitivity of the neuronal differentiated cells to BoNT which couldbe achieved by this differentiation medium was about 1 to 2 pM. In thenext step, it has been found that the addition of retinoic acid resultedin only a small increase in sensitivity to BoNT of the neuronaldifferentiated cells. In contrast, a significantly improved sensitivityby a factor of about 7 to 10 could be achieved by the use of neurobasalmedium. For example, a EC50 of 0.22 picomolar (2.18×10⁻¹³ mol/1) hasbeen found for SiMa cells when a differentiation medium comprisingneurobasal medium+2% B27 supplement+1% GlutaMAX+3 micromolar retinoicacid (RA) has been used, in the method of the invention. Further, itcould be shown that retinoic acid was not essential in thedifferentiation medium, for the improved sensitivity of the neuronaldifferentiated cells as long as neurobasal medium was included.Unexpectedly, the inventors could demonstrate that an improvedsensitivity could even be achieved by using the differentiation medium(comprising MEM+N2 supplement+B27 supplement) described in the art whichthey had used at the beginning of the series of experiments if onlydiluted to an osmolality of 225 mOsm/kg. In contrast, the non-diluteddifferentiation medium containing the mentioned components had anosmolality of 300 mOsm/kg. Accordingly, the increased sensitivity toBoNT of the neurotoxin-sensitive, neuronal differentiated cells producedby the methods of the invention is a result of the low osmolality of thedifferentiation medium. In addition to improved sensitivity, said noveldifferentiation protocol of the invention showed higher precision androbustness of testing the biological activity of neurotoxinpolypeptides, in comparison to methods known in the art.

As used herein, the singular forms “a”, “an” and “the” include bothsingular and plural reference unless the context clearly dictatesotherwise. By way of example, “a cell” refers to one or more than onecell.

As used herein, the term “about” when qualifying a value of a stateditem, number, percentage, or term refers to a range of plus or minus 10percent, 9 percent, 8 percent, 7 percent, 6 percent, 5 percent, 4percent, 3 percent, 2 percent or 1 percent of the value of the stateditem, number, percentage, or term. Preferred is a range of plus or minus10 percent.

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonyms with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps. Evidently, the term“comprising” encompasses the term “consisting of”. More specifically,the term “comprise” as used herein means that the claim encompasses allthe listed elements or method steps, but may also include additional,unnamed elements or method steps. For example, a method comprising stepsa), b) and c) encompasses, in its narrowest sense, a method whichconsists of steps a), b) and c). The phrase “consisting of” means thatthe composition (or device, or method) has the recited elements (orsteps) and no more. In contrast, the term “comprises” can encompass alsoa method including further steps, e.g., steps d) and e), in addition tosteps a), b) and c).

In case numerical ranges are used herein such as “retinoic acid in aconcentration between 0.1 and 0.5 micromolar”, the range includes notonly 0.1 and 0.5 micromolar, but also any numerical value in between 0.1and 0.5 micromolar, for example, 0.2, 0.3 and 0.4 micromolar.

The term “in vitro” as used herein denotes outside, or external to, theanimal or human body. The term “in vitro” as used herein should beunderstood to include “ex vivo”. The term “ex vivo” typically refers totissues or cells removed from an animal or human body and maintained orpropagated outside the body, e.g., in a culture vessel. The term “invivo” as used herein denotes inside, or internal to, the animal or humanbody.

The term “neurotoxin-sensitive cell” as used herein means a cell whichis susceptible to a neurotoxin polypeptide exhibiting the biologicalproperties characteristic for a neurotoxin polypeptide, namely, (a)receptor binding, (b) internalization, (c) translocation across theendosomal membrane into the cytosol, and/or (d) endoproteolytic cleavageof proteins involved in synaptic vesicle membrane fusion. Accordingly, a“neurotoxin-sensitive cell” as referred to herein is susceptible toneurotoxin intoxication. More specifically, “susceptible to neurotoxinintoxication” as denoted herein means a cell that can undergo theoverall cellular mechanisms whereby a neurotoxin polypeptide (e.g.,BoNT/A) cleaves a neurotoxin substrate (e.g., the BoNT/A substrateSNAP-25) and encompasses the binding of the neurotoxin to itscorresponding receptor (e.g., binding of BoNT/A to BoNT/A receptor), theinternalization of the neurotoxin/receptor complex, the translocation ofthe neurotoxin light chain from an intracellular vesicle into thecytoplasm, and the proteolytic cleavage of the neurotoxin substrate.Assays for determining the biological activity of a neurotoxinpolypeptide are well known in the art and also described elsewhereherein (see, e.g., Pellett et al., Withemarsh et al. ToxicologicalSciences 126(2), 426-435 (2012), WO 2010/105234 A1) As appreciated bythose skilled in the art, the neurotoxin-sensitive cell is preferablyable to first uptake a neurotoxin and then undergoes the overallcellular mechanisms listed above. A neurotoxin-sensitive cell as usedherein can uptake, e.g., about 100 nanomolar (nM), about 10 nM, about 1nM, about 500 picomolar (pM), about 400 pM, about 300 pM, about 200 pM,about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about50 pM, about 40 pM, about 30 pM, about 20 pM, about 10 pM, about 9 pM,about 8 pM, about 7 pM, about 6 pM, about 5 pM, about 4 pM, about 3 pM,about 2 pM, about 1 pM, about 0.5 pM, or about 0.1 pM of neurotoxinpolypeptide or even less than one of the indicated values. EC50 valuesabove 100 pM have been reported in the literature. By definition, a cellsusceptible to neurotoxin intoxication must express, or be engineered toexpress, at least one neurotoxin receptor and at least one neurotoxinsubstrate. Receptors and substrates for neurotoxins are described in theart. Accordingly, said cell is preferably susceptible to a biologicallyactive or mature neurotoxin polypeptide as defined herein. The term“neurotoxin-sensitive cell” as used herein comprises a cell or a cellline, for example, an isolated, primary cell or a cell line thereof or acell of an established cell line or an established cell line, preferablya neuroblastoma cell or neuroblastoma cell line as defined herein.Preferably, the “neurotoxin-sensitive cell” as used herein issusceptible to neurotoxin intoxication by, e.g., about 1 nM or less, 500pM or less, about 400 pM or less, about 300 pM or less, about 200 pM orless, about 100 pM or less, about 90 pM or less, about 80 pM or less,about 70 pM or less, about 60 pM or less, about 50 pM or less, about 40pM or less, about 30 pM or less, about 20 pM or less, about 10 pM orless, about 9 pM or less, about 8 pM or less, about 7 pM or less, about6 pM or less, about 5 pM or less, about 4 pM or less, about 3 pM orless, about 2 pM or less, about 1 pM or less, about 0.9 pM or less,about 0.8 pM or less, about 0.7 pM or less, about 0.6 pM or less, about0.5 pM or less, about 0.4 pM or less, about 0.3 pM or less, about 0.2 pMor less, or even about 0.1 pM or less. For example, an extremely lowEC50 value of less than 0.1 pM has been reached for SiMa cells and anEC50 of less than 1 pM for P19 cells, by using the novel differentiationmethod of the present invention. As known in the art, the “half maximaleffective concentration (EC50)” refers to the concentration of a drug,antibody or toxicant which induces a response halfway between thebaseline and maximum after some specified exposure time. It is commonlyused as a measure of drug's potency. The EC50 of a graded dose responsecurve therefore represents the concentration of a compound where 50% ofits maximal effect is observed. The EC50 of a quantal dose responsecurve represents the concentration of a compound where 50% of thepopulation exhibits a response, after a specific exposure duration.

Methods for the identification of cells or cell lines susceptible toneurotoxin intoxication and/or having neurotoxin uptake capacity, i.e.neurotoxin-sensitive cells as defined herein, are known in the art; see,e.g. US 2012/0122128 A1. The biological activity of the neurotoxinpolypeptides, in an aspect, results from all of the aforementionedbiological properties. Only a few cell-based assays with sufficient highsensitivity towards neurotoxins which can be used for the determinationof the biological activity of a neurotoxin have been described in theprior art so far, as indicated elsewhere herein. In vivo assays forassessing the biological activity of neurotoxins include, for example,the already mentioned mouse LD₅₀ assay and the ex vivo mousehemidiaphragm assay as described by Pearce et al. and Dressier et al.;see Pearce 1994, Toxicol. Appl. Pharmacol. 128: 69-77 and Dressier 2005,Mov. Disord. 20:1617-1619. The methods of the invention provide for asimple, reliable, and robust cell-based test system with increasedsensitivity to neurotoxin polypeptides, in comparison to the cellulartest systems described in the art which require complex differentiationprotocols. Accordingly, the methods of the invention provide for animproved alternative to the cellular test systems or animal tests of theart for determining the biological activity of neurotoxins. As known tothose skilled in the art, the biological activity of neurotoxins iscommonly expressed in Mouse Units (MU). One MU is the amount ofneurotoxic component, which kills 50% of a specified mouse populationafter intraperitoneal injection, i.e. the mouse i.p. LD 50.

The terms “differentiation”, “differentiating” or “differentiated” asused herein denote the process by which an unspecialized or a relativelyless specialized cell becomes relatively more specialized. In thecontext of cell ontogeny, the adjective “differentiated” is a relativeterm. Hence, a “differentiated cell” is a cell that has progressedfurther down a certain developmental pathway than the cell it is beingcompared with. A differentiated cell may, for example, be a terminallydifferentiated cell, i.e., a fully specialized cell that takes upspecialized functions in various tissues and organs of an organism, andwhich may but need not be post-mitotic. In another example, adifferentiated cell may also be a progenitor cell within adifferentiation lineage, which can further proliferate and/ordifferentiate. Similarly, a cell is “relatively more specialized” if ithas progressed further down a certain developmental pathway than thecell it is being compared with, wherein the latter is thereforeconsidered “unspecialized” or “relatively less specialized”. Arelatively more specialized cell may differ from the unspecialized orrelatively less specialized cell in one or more demonstrable phenotypiccharacteristics, such as, for example, the presence, absence or level ofexpression of particular cellular components or products, e.g., RNA,proteins, specific cellular markers or other substances, activity ofcertain biochemical pathways, morphological appearance, proliferationcapacity and/or kinetics, differentiation potential and/or response todifferentiation signals, etc., wherein such characteristics signify theprogression of the relatively more specialized cell further along thesaid developmental pathway.

The term “neuronal differentiated cell” as used herein means a cellwhich has reached the final neuronal differentiation status. Forexample, the murine embryonic carcinoma cells P19 cells which have beenused in the method of the present invention differentiate first toneuroprogenitor cells before they further differentiate to neurons. Theneurodifferentiation process can be followed, e.g., phenotypically (byphase contrast microscopy) and/or by the expression of neuronaldifferentiation markers; see, e.g., Babuska et al. (2010), PragueMedical Report 111, 289-299 or Migliore and Shepherd, Nature ReviewsNeuroscience 6, 810-818 (2005). Assays which can be used for thedetermination of the expression of said neuronal differentiation markersinclude, for example, PCR, RT-PCR, Northern blot, Western blot or Dotblot, immunoprecipitation analysis, enzyme-linked immunosorbent analysis(ELISA) or FACS analysis which are known in the art; see, e.g.,Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, third edition, 2001. Assays for testing the furthercharacteristics of neuronal differentiated cells are also known in theart.

The term “tumor cells which are able to differentiate into neuronalcells” as used herein means, for example, neuroblastoma cells, embryoniccarcinoma cells, teratocarcinoma cells, neural hybrid cells (e.g.neuron×glioblastoma cells), or fibroblastoma cells. The methods of theinvention have been exemplified using mouse and human tumor cells, i.e.P19 (murine embryonic carcinoma) and SiMa (human neuroblastoma) cells,respectively.

The term “neuroblastoma” as used herein means a cancer that developsfrom nerve cells found in several areas of the body. Neuroblastoma mostcommonly arises in and around the adrenal glands, which have similarorigins to nerve cells and sit atop the kidneys. However, neuroblastomacan also develop in other areas of the abdomen and in the chest, neckand pelvis, where groups of nerve cells exist. The term “neuroblastomacell” as used herein comprises one or more neuroblastoma cells which areneurotoxin-sensitive and capable of differentiating to neuronal cells.The neuroblastoma cell can be a primary neuroblastoma cell or a primaryneuroblastoma cell line. Encompassed by said term are also establishedneuroblastoma cells or cell lines. The neuroblastoma cell can be amammalian neuroblastoma cell, for example, a rodent neuroblastoma cellsuch as a rat or mouse neuroblastoma cell, but also a monkeyneuroblastoma cell, such as a rhesus, macaque, or cynomolgusneuroblastoma cell or a primate neuroblastoma cell such as a chimpanzeeneuroblastoma cell or, preferably, a human neuroblastoma cell. Examplesof established neuroblastoma cell lines encompass, e.g., Neuro-2a (mouseneuroblastoma), Kelly (human neuroblastoma), SH-SY5Y (humanneuroblastoma) or SiMa (human neuroblastoma). Human neuroblastoma cellsare preferably used in the methods of the invention in order to generateneurotoxin-sensitive, neuronal differentiated cells. More preferably,the neuroblastoma cell as defined herein is a SiMa cell or SiMa cellline. This is because SiMa cells are easy to transfer, in aBoNT-sensitive form. In addition, they have high sensitivity to BoNT.Moreover, the differentiation protocol for SiMa cells is simple andrather short, in comparison to other neuroblastoma cells or cell lines.The SiMa cells as used in the method of the invention can be parentalSiMa cells or (sub)clones derived therefrom.

In step a) of the aforementioned method of the invention, “cultivating”means cultivation of the tumor cells which are able to differentiateinto neuronal cells as defined herein in a cell culture medium underconditions and for a time which primes said tumor cells for neuronaldifferentiation. The term “priming” as used in cellular differentiationprocesses, is known in the art; see, e.g. Khoo et al., PLOS ONE 6(5):e19025, (2011); Steindler, ILAR Journal 48(4), 323-338 (2007); Vazey andConnor, Stem Cell Research & Therapy 1: 41 (2010). “Priming the tumorcells for neuronal differentiation” as used herein denotes that thetumor cells as defined herein are induced for neuronal differentiationby the indicated cell culture conditions, time and cell culture medium.Said priming of the tumor cells for neuronal differentiation can becarried out, for example, by cultivation in an appropriate cell culturemedium, and, optionally, by reduction of serum in the cell culturemedium and/or the addition of retinoic acid. As known to those skilledin the art, a differentiation status of the tumor cells in which saidtumor cells are “induced for neuronal differentiation” or “primed forneuronal differentiation” is characterized by, e.g., the expression ofneuronal induction markers as set forth elsewhere herein. As evident tothose skilled in the art, said “primed” or “induced” differentiationstatus of the tumor cells does, however, not yet represent the finalneuronal differentiation status of the tumor cells which is defined bythe characteristics as set forth elsewhere herein, but a previousdifferentiation step in the neuronal differentiation lineage. Only afterhaving carried out step b) of the differentiation method of theinvention, the tumor cells are finally differentiated, i.e. neuronaldifferentiated cells as specified herein. Cultivation of the tumor cellsas defined herein in a culture medium under conditions and for a timewhich primes said tumor cells for neuronal differentiation meanscultivating said tumor cells in an appropriate cell culture medium for12 hours to 7 days at 37° C., preferably 24 hours to 6 days, morepreferably 36 hours to 5 days and is known in the art. Appropriate cellculture media which can be used for priming said tumor cells forneuronal differentiation (also referred to herein as “priming medium”)include, for instance, OptiMEM, MEMalpha, RPMI-1640, Minimal essentialmedium (MEM), Ham's F12 medium, Dulbeccos modified Eagle's Medium (DMEM)or DMEM:F12 (1:1). The priming medium can comprise further componentsknown in the art, such as serum (e.g. FBS), NEAA, retinoic acid, growthfactors (such as NGF or FGF), vitamins, fatty acids, hormones and/orantibiotics. An appropriate cell culture medium for priming SiMa cellsfor neuronal differentiation comprises, for example, 80 to 98.6%OptiMEM, 1 to 7.5% FBS, 0.2 to 5% B27 supplement, 0.2 to 5% N2supplement, and, optionally, 0.1 to 2.5% non-essential amino acids(NEAA). Advantageously, it has been found by the present inventors thata cell culture medium comprising 92.5% OPTI-MEM®, 5% FBS, 1%non-essential amino acids, 1% B-27 supplement and 0.5% N-2 supplement isparticularly suitable for the priming of SiMa cells, as shown in thefollowing Example. For priming SiMa cells for neuronal differentiation,the preferred cultivation period is 12 hours to 5 days. Preferably, thepriming of SiMa cells is carried out under cell culture conditions asshown, in the following Example. For priming of P19 cells, for example,90 to 99.8% MEM-alpha, 0.2 to 10% FBS, and 0.001 to 10 micromolar (μM)retinoic acid (RA), preferably 0.1 μM RA, can be utilized. Here, thecultivation period is preferably 3 days to 7 days.

The term “differentiation medium” as used in step b) of the method forthe generation of neurotoxin-sensitive, neuronal differentiated cells ofthe invention is a cell culture medium having an osmolality of 100 to270 mOsm/kg. Preferably, the osmolality of the differentiation medium isbetween 120 to 250 mOsm/kg, more preferably between 150 and 240 mOsm/kg,even more preferably between 180 mOsm/kg and 230 mOsm/kg, mostpreferably between 200 and 225 mOsm/kg and most preferred about 225mOsm/kg. As set forth elsewhere herein and in the following Example, thepresent inventors have surprisingly found that the low osmolality of thedifferentiation medium is responsible for the increased sensitivity toBoNT of the neurotoxin-sensitive, neuronal differentiated cells producedby the method of the invention. As a differentiation medium, forexample, neurobasal medium, MEM, DMEM:F12 or any basal medium known inthe art can be used if adapted to the appropriate osmolality, e.g., byreduction of the NaCl concentration in the media formulation or bydilution of the media with water. Further, the differentiation mediumcomprises at least one supplement. The supplement can be (i) B27supplement, (ii) N2 supplement, (iii) NS21 supplement, or (iv)combinations thereof. For example, B27 supplement can be used incombination with N2 supplement. B27 supplement is known in the art andgenerally used for growth and maintenance of neurons. Furthermore, B27supplement is commercially available, e.g., from Life Technologies.Typically, B27 supplement can be used in a concentration of 0.2% to 5%in the method of the invention. N2 supplement (which can be obtainedfrom, e.g., Life Technologies) is a chemically defined, serum-freesupplement based on Bottenstein's N-1 formulation. N2 Supplement isrecommended for growth and expression of neuroblastomas as well aspost-mitotic neurons in primary cultures from both the peripheralnervous system (PNS) and the central nervous system (CNS). N2 supplementcan be used in a concentration of 0.2% to 5% in the method of theinvention. NS21 supplement is a re-defined and modified B27 supplementin which 21 different ingredients have been used for neuronal cultures,as described, for example, in the publication by Chen et al. (2008),Journal of Neuroscience Methods 171, 239-247. Typically, NS21 supplementis used in a concentration of 0.2% to 5% in the method of the invention.Further ingredients used in the above-referenced differentiation mediumcomprise Glutamine or GlutaMax, one or more antibiotic agents, NEAA, FBS(0.1 to 20%), GT1b (0.1 to 300 μM), retinoic acid (RA) (1 nanomolar (nM)to 300 μM), growth factors (such as 100 ng/ml NGF, 40 ng/ml BDNF, 40ng/ml CNTF, 5 ng/ml LIF, 5 ng/ml GDNF, TGF or FGF) or furtherdifferentiation factors known in the art (1% DMSO, 1 mM Dibutyryl cAMP,1 mM Butyrate, 5 μg/ml celecoxib, Y-27632, SB431542, sonic hedgehog(SHH) etc.). The cells of step b) of this method of the invention arecultivated in said differentiation medium preferably for at least 3 daysin order to obtain neurotoxin-sensitive, neuronal differentiated cells.Cultivation can also be longer, for example, for at least 3.5 days, 4days, 4.5 days, 5 days, 6 days, 7 days (1 week), 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days (2 weeks), 15 days, 16 days, 17days, 18 days, 19 days, 20 days, 21 days (3 weeks) or even longer.Preferred is cultivation of P19 tumor cells for 2 to 3 weeks, andcultivation of SiMa cells for 3 days to 7 days. Preferreddifferentiation media and cell culture conditions which allow for theneuronal differentiation are shown in the following Example. Preferably,said neuronal differentiated cells represent finally differentiatedneuronal cells as defined elsewhere herein.

The term “osmolality” as used herein means the measure of soluteconcentration, which can be defined as the number of osmoles (Osm) ofsolute per kilogram (kg) of solution (osmol/kg or Osm/kg). Theosmolality of a solution is usually expressed as Osm/kg. Osmolalitymeasures the number of osmoles of solute particles per unit mass ofsolution. Osmolarity is a measure of the osmoles of solute per liter ofsolvent (osmol/l or Osm/l). Osmolality and osmolarity can be determinedon an osmometer.

In a further aspect, the aforementioned method of the invention cancomprise additional steps. For example, said additional steps canencompass steps for determining the biological activity of a neurotoxinpolypeptide as defined herein. To this end, the neurotoxin-sensitive,neuronal differentiated cells obtained or obtainable by the methods ofthe invention are first brought in contact with a neurotoxinpolypeptide. The term “contacting” as used in accordance with themethods of the invention refers to bringing the aforementioned cells andthe neurotoxin in physical proximity as to allow physical and/orchemical and/or biological interaction. The neurotoxin can be comprisedby a sample, preferably a biological sample such as a cell, cell lysate,blood, plasma, serum or lymph fluid. Suitable conditions which allow forspecific interaction are well known to the skilled worker. Saidconditions will depend on the cells and neurotoxins to be applied in themethods of the present invention, and can be adapted by the skilledartisan without further ado. Moreover, a time being sufficient to allowinteraction can also be determined by the skilled worker by routineexperimentation. For example, a specific amount of an isolated orrecombinant neurotoxin polypeptide or a variant thereof as definedherein or a sample comprising a neurotoxin polypeptide can be added tothe neurotoxin-sensitive, neuronal differentiated cells. Thereafter, thecells are incubated with the neurotoxin polypeptide for at least 24 h,preferably 48 h, more preferably for 72 h under conditions which allowfor the neurotoxin polypeptide to exert its biological activity.“Conditions which allow for the neurotoxin polypeptide to exert itsbiological activity” as used herein are known in the art. Subsequently,the cells are stopped, for example by the addition of lysis buffer, andthe biological activity of the neurotoxin polypeptide is determined asshown, for instance, in the following Example.

The term “neurotoxin”, “neurotoxin polypeptide” or “neurotoxin protein”as used in the present invention refers to the seven distinct serotypesof Botulinum neurotoxins, i.e. BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E,BoNT/F, BoNT/G, and to Tetanus Neurotoxin (TeNT), and variants thereofas defined herein. The corresponding nucleic acid and amino acidsequences are known in the art; see, e.g., Uniprot or TREMBL sequencedatabase. Preferably, BoNT/A is used in the methods of the invention (WO2009/114748). The corresponding receptors and substrates for saidneurotoxins have been referred to elsewhere herein. The neurotoxinpolypeptide can be a naturally occurring neurotoxin or a non-naturallyoccurring neurotoxin. A naturally occurring neurotoxin polypeptide isproduced by a naturally occurring process, including, for example,isoforms produced from a post-translational modification, analternatively-spliced transcript or a spontaneous mutation and subtypes.For instance, BoNT/A subtypes are BoNT/A1 subtype, BoNT/A2 subtype,BoNT/A3 subtype, BoNT/A4 subtype or BoNT/A5 subtype. A naturallyoccurring neurotoxin polypeptide includes the above-referenced sequencesin which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or moreamino acid residues are added, substituted or deleted. Commerciallyavailable pharmaceutical compositions which comprise anaturally-occurring BoNT/A have already been mentioned in theintroductory part. A non-naturally occurring neurotoxin polypeptidemeans any neurotoxin polypeptide whose structure was modified with theaid of human manipulation, including, for example, a neurotoxinpolypeptide with an altered amino acid sequence produced by geneticengineering using random mutagenesis or rational design and a neurotoxinpolypeptide generated by chemical synthesis. Such non-naturallyoccurring neurotoxin polypeptides have been described in the art.

In another aspect of the invention, the neurotoxin polypeptide has anamino acid sequence being at least 40%, at least 50%, at least 60%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98% or at least 99% identical to the amino acidsequence of BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G, orTetanus neurotoxin as defined herein. Identical as used in the presentinvention refers to sequence identity of amino acid sequences whereinthe sequences are aligned so that the highest order match is obtained.This can be achieved by using published techniques or methods codifiedin computer programs such as, for example, BLASTP, BLASTN, FASTA,Altschul 1990, J. Mol. Biol. 215, 403. The percent identity values are,in one aspect, calculated over the entire amino acid sequence. A seriesof programs based on a variety of algorithms is available to the skilledworker for comparing different sequences. In this context, thealgorithms of Needleman and Wunsch or Smith and Waterman giveparticularly reliable results. To carry out the sequence alignments, theprogram PileUp (1987, J. Mol. Evolution 25, 351; Higgins 1989 CABIOS 5,151) or the programs Gap and BestFit (Needleman and Wunsch 1970, J MolBiol 48; 443; Smith and Waterman 1981, Adv. Appl. Math. 2, 482), whichare part of the GCG software packet (Genetics Computer Group 1991, 575Science Drive, Madison, Wis., USA 53711), are to be used. The sequenceidentity values recited above in percent (%) are to be determined, inone aspect of the invention, using the program GAP over the entiresequence region with the following settings: Gap Weight: 50, LengthWeight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which,unless otherwise specified, shall always be used as standard settingsfor sequence alignments. It will be understood that the aforementionedvariants shall, in an aspect of the invention, retain, at least one ofthe biological properties of neurotoxins and, in an aspect, all of thebiological properties of a neurotoxin polypeptide recited herein. In afurther aspect, the variants can be neurotoxins having improved oraltered biological properties, e.g., they may comprise cleavage siteswhich are improved for enzyme recognition or may be improved forreceptor binding or any other property specified above.

The neurotoxins referred to herein, in principle, comprise an N-terminallight chain and a C-terminal heavy chain. The neurotoxins are producedas single chain precursor molecules, referred to as “unprocessedneurotoxin polypeptides”. As a result of the subsequent processing,“processed neurotoxin polypeptide” is obtained. The said processedneurotoxin polypeptide exhibits the biological properties characteristicfor a neurotoxin, namely, (a) receptor binding, (b) internalization, (c)translocation across the endosomal membrane into the cytosol, and/or (d)endoproteolytic cleavage of proteins involved in synaptic vesiclemembrane fusion. Therefore, the processed neurotoxin polypeptide isreferred to as biologically active or mature neurotoxin polypeptide. Thebiological activity of the neurotoxin polypeptides, in an aspect,results from all of the aforementioned biological properties, as setforth elsewhere herein.

In another aspect, the neurotoxin polypeptide in accordance with themethod of the invention may be a chimeric molecule. Said chimericmolecule, in one aspect, may have single domains substituted.Accordingly, in another aspect, the portion of the neurotoxin heavychain is replaced by a portion of an Fc domain of an antibody.

The term “amount” as used herein encompasses the absolute amount of,e.g., a neurotoxin polypeptide, the relative amount or the concentrationof the said polypeptide as well as any value or parameter whichcorrelates thereto or can be derived there from.

The term “determining the amount” of, e.g., a neurotoxin polypeptiderelates to measuring the absolute amount, relative amount orconcentration of, e.g., the neurotoxin polypeptide in a quantitative orsemi-quantitative manner. Suitable measures for detection are well knownto those skilled in the art. It will be understood that thedetermination of the amount of neurotoxin polypeptides, in an aspect,also requires calibration of the method by applying standard solutionswith predefined amounts of neurotoxin polypeptides. It is well known tothose skilled in the art how to carry out such a calibration.

The term “determining the biological activity of a neurotoxinpolypeptide” as used herein means measuring the biological activity of aneurotoxin protein, namely, (a) receptor binding, (b) internalization,(c) translocation across the endosomal membrane into the cytosol, and/or(d) endoproteolytic cleavage of proteins involved in synaptic vesiclemembrane fusion. More specifically, the overall cellular mechanismswhereby a neurotoxin (e.g., BoNT/A) cleaves a neurotoxin substrate(e.g., SNAP-25) encompasses the binding of the neurotoxin to itscorresponding receptor (e.g., binding of BoNT/A to BoNT/A receptor), theinternalization of the neurotoxin/receptor complex, the translocation ofthe neurotoxin light chain from an intracellular vesicle into thecytoplasm and the proteolytic cleavage of the neurotoxin substrate. Invitro and in vivo assays for determining the biological activity of aneurotoxin polypeptide are well known in the art and have been mentionedelsewhere herein (see, e.g., Pellett et al., Withemarsh et al Toxicol.Sciences 126(2), 426-435 (2012), WO 2010/105234 A1).

As used herein, the term “sensitivity to neurotoxin (polypeptide)activity” refers to the lowest dose that an assay can measureconsistently above the signal detected by a non-treatment control orbackground signal.

In one aspect of the method of the invention, the differentiation mediumas used in step b) of the aforementioned method of the invention furthercomprises retinoic acid. Preferably, said retinoic acid is present inthe neurobasal medium in a concentration of between 0.01 micromolar (μM)and 300 μM. More preferably, retinoic acid is present in a concentrationof between 0.1 and 0.5 μM retinoic acid for P19 tumor cells, i.e. in aconcentration of 0.1, 0.2, 0.3, 0.4 or 0.5 μM retinoic acid; and between1 and 5 μM for SiMa cells, i.e. in a concentration of 1, 2, 3, 4, or 5μM, most preferably in a concentration of 3 μM. Retinoic acid is a knownneural inducer which is able to induce differentiation of neuronalcells, including neuroblastoma cells. The addition of low concentrationsof retinoic acid into the differentiation medium used in step b) of themethod of the invention has been found to enhance advantageously thesensitivity of the tumor cells to neurotoxin polypeptides.

In a further aspect of the method of the invention, said differentiationmedium in step b) further comprises an antibiotic agent and/or acytostatic agent which inhibits growth of non-neuronal cells. Asantibiotic agent, for example, penicillin-streptomycin can be used. As acytostatic agent, for instance, cytosine-1-β-D-arabinofuranoside (AraC)can be used. The addition of an antibiotic agent prevents the growth ofbacteria in the cell culture medium, whereas the cytostatic agentinhibits cell growth and multiplication of non-tumor/non-neuronal cellswhich could otherwise overgrow the tumor cells in the differentiationmethod of the present invention.

In still another aspect of the method of the invention, saiddifferentiation medium in step b) further comprises GT1b. GT1b is aganglioside which binds to neurotoxin and potentially mediates theselectivity of neurotoxins for neurons. Accordingly, GT1b can be used asan enhancer for the BoNT uptake into the tumor cells in the methods ofthe invention. Preferably, said GT1b is present in a concentration ofbetween 25 and 75 μM, i.e. in a concentration of 25 μM, 30 μM, 35 μM, 40μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM or 75 μM, more preferablyin a concentration of 50 μM.

In yet another aspect of the method of the invention, step a) of saidmethod of the invention comprises cell culture conditions comprisingreduction of serum from the (cell) culture medium and/or addition ofretinoic acid. “Reduction of serum from the culture medium” as usedherein means, for example, stepwise reduction of serum, e.g., at aninterval of 2 to 5 days, from 10% to 5% to 2.5% to 1% to 0%.Alternatively, serum-containing cell culture medium can be replaced byserum-free cell culture medium, in one step. The removal of growthfactors contained in the serum allows for the differentiation of thetumor cells as defined herein. In addition or alternatively, retinoicacid can be added to the culture medium in low concentrations as setforth elsewhere herein.

In some aspects of the method of the invention, said tumor cells whichare able to differentiate into neuronal cells are SiMa cells available,e.g., from DSMZ (German collection of Microorganisms and Cell cultures)under the ACC deposit number: 164. The SiMa cell line DSMZ ACC 164 isalso known as parental SiMa cell line. SiMa cells as used in the methodsof the invention can be parental SiMa cells or (sub)clones thereof. Suchsubclones are also known in the art; see, e.g., WO 2010/105234, U.S.Pat. No. 8,476,068 B2 or Ester Fernéandez-Salas, Joanne Wang, YaniraMolina, Jeremy B. Nelson, Birgitte P. S. Jacky, K. Roger Aoki—PloSOne;2012 7(11) e49516.

SiMa cells can be cultivated according to the protocol of the DMSZ. Inan aspect, cultivating SiMa cells in a cell culture medium underconditions and for a time which primes said SiMa cells for neuronaldifferentiation in step a) of the method of the invention comprisescultivation of said SiMa cells in a cell culture (or growth) mediumcomprising at least OptiMEM, FBS, B27 supplement, N2 supplement and,optionally NEAA, for at least 12 hours (h), at least 24 hours orpreferably at least 36 hours at 37° C.

In an aspect of the method of the invention, the culture or primingmedium for SiMa cells in step a) comprises 80% to 98.8% OptiMEM, 1 to10% FBS, 0.2 to 5% B27 supplement and/or 0.2 to 5% N2 supplement.Optionally, said culture medium for SiMa cells further comprisesnon-essential amino acids and/or an antibiotic and/or retinoic acid.

Preferably, the culture or priming medium for SiMa cells in step a)comprises 92.5% OptiMEM, 5% FBS, 1% B27 supplement, 0.5% N2 supplementand 1% NEAA.

In an aspect of the method of the invention, the differentiation mediumin step b) comprises a medium having an osmolality of 100 to 270mOsm/kg. Preferably, said medium is neurobasal medium used in aconcentration of 86 to 98.8%. The differentiation medium can furthercomprise one or more of the following ingredients: 0.2 to 5% B27supplement and/or 0.2 to 5% N2 supplement; 0.5 to 2% non-essential aminoacids (NEAA); 1 to 5 μM retinoic acid; 0.5 to 2% GlutaMAX and/or 25 to75 μM GT1b.

Preferably, the differentiation medium in step b) comprises 97%neurobasal medium, 2% B27 supplement, 1% GlutaMAX, and 3 μM retinoicacid.

In an aspect of the method of the invention, the SiMa cells arecultivated in step a) for at least 36 hours.

In an aspect of the method of the invention, the SiMa cells arecultivated in step a) on tissue culture dishes which are coated with atleast one compound selected from the group consisting of: poly-L-lysine,poly-D-lysine, collagen, laminins, and gelatine. Coating of cell culturedishes is well known to those skilled in the art. Preferably, the tissueculture dishes are coated with poly-L-lysine.

The present invention provides for a novel differentiation protocol withwhich a significant increase in the sensitivity of SiMa cells toneurotoxin polypeptides can be achieved. In addition, said test systemis more precise and robust than tests for the biological activity ofneurotoxin polypeptide described in the art. In a first step for primingthe SiMa cells for neuronal differentiation, the SiMa cells werecultivated in a novel priming medium comprising 92.5% Opti-MEM®, 5% FBS,1% B27 supplement, 0.5% N2 supplement and 1% NEAA. If applicable, one ormore antibiotic agents were also be added to the cell culture medium.The cells were then seeded on coated multi-well plates. Optimal resultshave been achieved with cell culture dishes which were coated withpoly-L-lysine. Alternatively, collagen, poly-D-lysine, laminins,gelatin, or combinations thereof can also be used for the coating of thecell culture dishes. After at least 36 h growth of the seeded cells inthe above-referenced growth culture or priming medium, said medium hasbeen substituted by six different differentiation media, in comparison.After three to seven days of differentiation in which thedifferentiation medium has been replaced by fresh differentiationmedium, if applicable, neurotoxin polypeptide (as exemplified by BoNT/A)was added to the differentiation medium. After further 75 h ofincubation, the cells were stopped and analyzed for biological activityof the added neurotoxin polypeptide. In comparison, differentiationmedia with low osmolality delivered the best results: The use of adifferentiation medium comprising neurobasal medium containing 2% B27supplement, 1% GlutaMAX and 3 μM retinoic acid provided EC50 values of0.22 pM. Said differentiation medium had an osmolality of about 225mOsm/kg. A similar result of an EC50 value of 0.24 pM was found for adifferentiation medium comprising MEM, 2% B27 supplement, 1% N2supplement, and 3 μM retinoic acid, diluted to about 225 mOsm/kg. Thecomparatively simple differentiation protocol of the present inventionallows for high reproducibility and high sensitivity (EC50 below 0.3 pM;LLOD below 0.1 pM), which allows for high dilutions of the sample to beanalyzed. Accordingly, the sensitivity of SiMa cells which have beengenerated by the differentiation method of the present invention ispreferably, less than 10 pM, less than 5 pM, less than 2 pM, less than 1pM, less than 0.5 pM or even less than 0.3 pM.

In other aspects of the method of the invention, said tumor cells whichare able to differentiate into neuronal cells are P19 cells which can beobtained, e.g., from DSMZ under the ACC deposit number: 316, from ATCCunder CRL-1825 or from ECACC under 95102107. P19 cells can be cultivatedaccording to the protocol of the DSMZ. In an aspect, cultivating the P19cells in a cell culture medium under conditions and for a time whichprimes said P19 cells for neuronal differentiation in step a) of themethod of the invention comprises cultivation of said P19 tumor cells onbacteriological petri dishes in a cell culture (or growth) mediumcomprising at least MEM-alpha and FBS for at least 3 days, 4 days orpreferably 5 days such that cell aggregates are formed. In an aspect,said cell culture medium for P19 cells comprises retinoic acid,preferably in a concentration of between 75 and 125 nM, more preferably100 nM. In an aspect, the cell culture medium comprises 95% MEM-alpha,5% FBS and 0.1 μM retinoic acid. Preferably, the P19 cell aggregates areharvested and isolated cells primed for neuronal differentiation aregenerated from the aggregates by protease treatment, e.g. trypsintreatment. In an aspect of the method of the invention, the P19 cells instep b) of the method of the invention are cultivated for 2 to 3 weeksin differentiation medium with low osmolality as defined herein,preferably neurobasal medium with an osmolality of about 225 mOsm/kg.

The present invention provides for a novel differentiation protocol forP19 cells with which a significant increase in the sensitivity of thecells towards BoNT could be achieved, as demonstrated in the followingExample. In the differentiation protocol of the invention, P19 cellswere cultured as described in the prior art. For priming the P19 cellsfor neuronal differentiation, the cells were seeded on petri dishes usedfor bacteria (i.e. no cell culture dishes) and 100 nM retinoic acid wasadded. After five days, the resulting cell aggregates were isolated andtrypsinized. Thereafter, the cells were seeded on multi-well plates andcultivated further. It has surprisingly been found by the presentinventors that the use of neurobasal medium (neurobasal mediumcomprising 2% B27 supplement, 1% N2 supplement and 1% GlutaMAX) as wellas the addition of low concentrations of retinoic acid (100 nM) couldincrease the sensitivity of the cells towards BoNT further. The growthof non-neuronal cells could be inhibited by the addition of a cytostaticdrug (for example, 5 μM AraC). Moreover, it has been found that theaddition of GT1b enhances the uptake of BoNT of the cells when GT1b isadded to the medium upon intoxication of the cells with BoNT. To thisend, for example, 50 μM GT1b has been used. After two to three weeks ofdifferentiation, in which the medium has been replaced if necessary,BoNT was added to the cell culture medium. After 72 h of incubation withBoNT, the cells were stopped and analyzed by methods shown in thefollowing Example.

As exemplified for two different cell lines, the present inventionprovides for a novel cell-based test system for the determination of thebiological activity of neurotoxins. Said cell-based test system of theinvention is extremely sensitive and robust and provides an alternativeto conventional animal tests. Furthermore, the cell-based test system ofthe invention is a simple differentiation protocol, in comparison tocell-based test systems described in the art and allows for highreproducibility and high sensitivity (EC50 below 0.1 pM for SiMa cells,and below 1 pM for P19 cells) which allows for high dilutions of theneurotoxin-containing samples to be analyzed. Such high dilutions of thesamples is extremely important as regards excipients or impuritiescontained in the sample to be analyzed in order to apply saidpotentially disturbing substances in concentrations as low as possible.Finally, the cell-based test system of the invention is more flexibleand economic than systems described in the prior art.

It is to be understood that the definitions and explanations of theterms made above apply mutatis mutandis for all aspects described inthis specification in the following except as otherwise indicated.

The invention further relates to a neurotoxin-sensitive, neuronaldifferentiated cell obtainable or obtained by the method of theinvention. Preferably, in case SiMa cells are used, said cells have anEC50 value of less than 10 pM, less than 5 pM, less than 2 pM, less than1 pM, less than 0.5 pM or even less than 0.3 pM. It is also preferredthat the sensitivity for Botulinum neurotoxins of neuronaldifferentiated SiMa cells generated by the differentiation method of theinvention is increased at least 1.5 fold, at least 2 fold, at least 3fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7fold, at least 8 fold, at least 9 fold or even at least 10 fold, incomparison to SiMa cells differentiated in a differentiation mediumhaving an osmolality of about 290 to 350 mOsm/kg which is used commonly,in neuronal differentiation processes described in the art. Similarresults could be achieved using P19 cells; see Example.

In addition, the invention provides for a method for determining theactivity of a neurotoxin polypeptide comprising the steps of:

-   -   a) contacting the neurotoxin-sensitive, neuronal differentiated        cells, preferably obtainable or obtained by the method of the        invention, with a neurotoxin polypeptide;

b) cultivating the neurotoxin-sensitive, neuronal differentiated cellsof step a) for 3 to 74 hours or preferably 72 hours under conditionswhich allow for the neurotoxin polypeptide to exert its biologicalactivity; and

-   -   c) determining the biological activity of the neurotoxin        polypeptide in the said cells after cultivation according to        step b).

Specifically, said method for determining the activity of a neurotoxinpolypeptide comprises the steps of:

-   -   a) cultivating tumor cells which are able to differentiate into        neuronal cells in a culture medium under conditions and for a        time which primes said tumor cells for neuronal differentiation;    -   b) cultivating the tumor cells primed for neuronal        differentiation of step a) in a differentiation medium having an        osmolality of 100 to 270 mOsm/kg, and comprising (i) B27        supplement and/or (ii) N2 supplement, for at least 3 days,        thereby obtaining neurotoxin-sensitive, neuronal differentiated        cells;    -   c) contacting the neurotoxin-sensitive, neuronal differentiated        cells of step b) with a neurotoxin polypeptide;    -   d) cultivating the neurotoxin-sensitive, neuronal differentiated        cells of step c) for 3 to 74 hours, or preferably 72 hours,        under conditions which allow for the neurotoxin polypeptide to        exert its biological activity; and    -   e) determining the biological activity of the neurotoxin        polypeptide in the said cells after cultivation according to        step d).        The determination of the biological activity of the neurotoxin        polypeptide can be carried out by methods described in the art        (see, e.g., Pellett et al., Withemarsh et al. Toxicological        Sciences 126(2), 426-435 (2012), WO 2010/105234 A1, WO        2009/114748, WO 2012/123370, WO 2013/131991). Preferably, the        tumor cells which are able to differentiate into neuronal cells        are SiMa cells, more preferably parental SiMa cells (DSMZ ACC        164). The neurotoxin polypeptide is preferably BoNT/A.

The invention pertains also to the use of a cell culture medium and/ordifferentiation medium as specified in the method of the invention forgenerating neurotoxin-sensitive, neuronal differentiated cells ex vivofrom neuronal differentiated cells, preferably neuroblastoma cells.

In a further aspect, the invention provides for a cell culture orpriming medium comprising 80 to 98.8% OptiMEM, 1 to 10% FBS, 0.2 to 5%B27 supplement and/or 0.2 to 5% N2 supplement. Alternatively, 0.2 to 5%NS21 supplement can be used. Preferably, said medium comprises 93.5%OptiMEM, 5% FBS, 1% B27 supplement and 0.5% N2 supplement. Said mediumis particularly useful for priming SiMa cells. Opti-MEM® reduced serummedia (Life Technologies) is a modification of Eagle's Minimum EssentialMedia, buffered with HEPES and sodium bicarbonate and supplemented withhypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elementsand growth factors. The culture medium can in an aspect further comprisean antibiotic agent as defined herein and/or non-essential amino acids(NEAA). Said culture medium of the invention is particularly useful instep a) of the method for generation of neurotoxin-sensitive, neuronaldifferentiated cells of the invention using SiMa cells.

Finally, the invention relates to a kit adapted for carrying out theaforementioned methods, said kit comprising a cell culture mediumcomprising 80 to 98.8% OptiMEM, 1 to 10% FBS, 0.2 to 5% B27 supplementand/or 0.2 to 5% N2 supplement and, optionally, non-essential aminoacids and/or an antibiotic, and SiMa cells. Preferably, said cellculture medium comprises 93.5% OptiMEM, 5% FBS, 1% B27 supplement and0.5% N2 supplement, and SiMa cells. The kit can further comprise adifferentiation medium as defined herein having an osmolality of 100 to270 mOsm/kg, preferably neurobasal medium, and at least one supplementas indicated herein, i.e. B27, N2 or NS21 supplement. Preferably, saiddifferentiation medium comprises 78 to 98.3% neurobasal medium, 1 to 10%FBS, 0.5 to 2% GlutaMAX, 0.2 to 5% B27 supplement, and/or 0.2 to 5% N2supplement. Further preferred media which can be used in the kit of theinvention are shown in the following Example.

It is to be understood that the kit of the present invention is to beused for practicing the methods referred to herein above. In one aspect,it is envisaged that all components are provided in a ready-to-usemanner for practicing the methods referred to above. In a furtheraspect, the kit contains instructions for carrying out the said methods.The instructions can be provided by a user manual in paper- orelectronic form. For example, the manual may comprise instructions forinterpreting the results obtained when carrying out the aforementionedmethods using the kit of the present invention.

All references cited in this specification are herewith incorporated byreference with respect to their entire disclosure content and thedisclosure content specifically mentioned in this specification.

Abbreviations as Used Herein:

OptiMEM: Opti-MEM® reduced serum media (Life Technologies) is amodification of Eagle's Minimum Essential Media, buffered with HEPES andsodium bicarbonate and supplemented with hypoxanthine, thymidine, sodiumpyruvate, L-glutamine, trace elements and growth factors. Most cellsgrown in serum-supplemented media can be transferred to Opti-MEM® with aminimum of 50% reduction in serum.

FBS: Fetal bovine serum is the most widely used serum-supplement for thein vitro cell culture of eukaryotic cells. FBS is commercially availablefrom many manufacturers, such as Sigma-Aldrich, Invitrogen, LifeTechnologies and others.

B27 supplement: B27 supplement is generally used for growth andmaintenance of neurons. B27 supplement is commercially available as 50×or 100× stock solutions, e.g., from Life Technologies.

N2 supplement: Commercially obtainable as 50× or 100× stock solutions,e.g., from Life Technologies. N2 supplement is recommended for growthand expression of neuroblastomas as well as post-mitotic neurons inprimary cultures from both the peripheral nervous system and the centralnervous system. N2 supplement can be used as a substitute forBottenstein's N1 formulation. N2 supplement can be used with neurobasalmedia supplemented with growth factors such as bFGF or EGF or used withDMEM.

NS21 supplement: NS21 supplement is a re-defined and modified B27supplement in which 21 different ingredients have been used for neuronalcultures, as described, for example, in the publication by Chen et al.(2008), Journal of Neuroscience Methods 171, 239-247.

NEAA: Non-essential amino acids cell culture supplement commerciallyavailable by many manufacturers such as Invitrogen or Cyagen is usuallyprovided as a stock solution containing several kinds of non-essentialamino acids such as glycine, L-alanine, L-asparagine, L-glutamic acid,L-aspartic acid, L-serine or L-proline. It is used as a supplement tocell culture media for optimized cell growth.

P19 cells: P19 cells are embryonic carcinoma cell lines derived from anembryo-derived teratocarcinoma in mice. The cell line is multipotentcells which can differentiate into all three germ layers cell types.Also, it is the most characterized embryonic carcinoma (EC) cell linethat can be specific induced into cardiac muscle cells and neuronalcells by different specific treatment. Exposing aggregated P19 cells todimethyl sulfoxide (DMSO) can let differentiate into cardiac andskeletal muscle. Also, exposing P19 cells to retinoic acid (RA) candifferentiate them into neuronal cells (McBurney & Rogers (1982), Dev.Biol 89(2):503-508; Rudnicki, et al. (1990) Dev. Biol 138(2):348-358).

SiMa cells: SiMa cells correspond to a neuroblastoma (Nb) cell line,carrying the major recurrent chromosome changes associated with poorprognosis Nb, including amplification of N-MYC by formation of doubleminutes (dmin), der(1)t(1; 17)(p35; q12) and der(22)t(17; 22)(q22; p13),and loss of chromosome 11, documented at both initiation and latepassage. In contrast to these cytogenetic stigmata of poor prognosis,analysis of catecholamine synthesis by high pressure liquidchromatography (HPLC) measurement revealed an advanced degree ofadrenergic differentiation with high rates of 3,4-Dihydroxyphenylalanine(DOPA), noradrenaline, homovanillic acid (HVA), and vanillylmandelicacid (VMA) production. Contrastingly advanced differentiation and poorprognostic genetic markers combine to render SiMa a unique instrumentfor investigating the pathology and therapy of Nb (Marini et al., CancerGenet. Cytogenet. (1999), 112:161-4).

MEM-alpha: Minimal Essential Medium Alpha (Invitrogen, LifeTechnologies)

Neurobasal medium: Neurobasal® Medium (Invitrogen, Life Technologies) isa basal medium that meets the special cell culture requirements ofpre-natal and embryonic neuronal cells when used with GIBCO® B-27®Supplement. Neurobasal® Medium can be used to grow neuronal cells fromhippocampus, cortex and other regions of the brain. Neurobasal® Mediumallows for both long and short term maintenance of homogeneouspopulations of neuronal cells without the need of an astrocyte feederlayer.

GlutaMax: GIBCO® GlutaMAX™ media is a cell culture media which containsa stabilized form dipeptide from L-glutamine, L-alanyl-L-glutamine, thatprevents degradation and ammonia build-up even during long-termcultures. Extremely stable in aqueous solution, the L-alanyl-L-glutaminedipeptide will not degrade into ammonia in storage or incubation likeL-glutamine.

The invention will now be illustrated by the following Example whichshall, however, not be construed as limiting the scope of the presentinvention.

EXAMPLE

The Influence of Differences Between Neurobasal and MEM Medium on theBotulinum Neurotoxin (BoNT) Sensitivity

i) SiMa parental cells were cultivated according to the protocol of theDSMZ (German collection of Microorganisms and Cell cultures). One daybefore plating the cells on 96-well plates, the medium was changed to92.5% OPTI-MEM® (Gibco by Life Technologies™ #51985)+5% FBS (PAA#A15-152)+1% non-essential amino acids (NEAA; Gibco by LifeTechnologies™ #11140-038)+1% B-27 supplement (Gibco by LifeTechnologies™ #17504-044)+0.5% N-2 supplement (Gibco by LifeTechnologies™ #17502-048) as “priming medium. Cells (30.000 cells/cm²)were plated on 96-well plates (TPP #92096) five days before addition ofBotulinum Neurotoxin (BoNT) in the above mentioned priming medium. 24 hafter plating the cells, the medium was exchanged for one of thefollowing differentiation media:

-   1. 97% Neurobasal (Gibco by Life Technologies™ #21103)+2% B-27    supplement (Gibco by Life Technologies™ #17504-044)+1% GlutaMAX™    (Gibco by Life Technologies™ #35050-038)+3 μM retinoic acid (RA;    Sigma-Aldrich #R2625)    or-   2. 96% MEM (Gibco by Life Technologies™ #42360)+2% B-27 supplement    (see above)+1% N-2 supplement (see above)+1% NEAA (see above)+3 μM    RA (see above)    or-   3. 96% MEM (see above)+2% B-27 supplement (see above)+1% N-2    supplement (see above)+1% NEAA (see above)+3 μM RA (see    above)+Vitamin B12+Fe+Zn (low)    or-   4. 96% MEM (see above)+2% B-27 supplement (see above)+1% N-2    supplement (see above)+1% NEAA (see above)+3 μM RA (see    above)+Vitamin B12+Fe+Zn (high)    or-   5. 96% MEM (see above)+2% B-27 supplement (see above)+1% N-2    supplement (see above)+1% NEAA (see above)+3 μM RA (see above);    diluted to 225 mOsm/kg    or-   6. 97% Neurobasal (see above)+2% B-27 supplement (see above)+1%    GlutaMAX™ (see above)+3 μM retinoic acid (see above)+Zn (high)

Four days after the change to differentiation medium, half of the mediumper well is exchanged for fresh medium which contains BoNT/A indifferent concentrations (serial 1:2 dilution in 11 steps starting from10 pM plus negative control without BoNT/A). 72 h after addition ofBoNT, the medium was aspirated and lysis buffer containing benzonase wasadded. The plate was incubated at room temperature (RT) and thenRoti-load 1 (Roth #K929.1) was added. Samples prepared as aforementionedcould be stored at a temperature below −70° C. The low temperature iscritical because SNAP-25 seems to be specifically degraded at highertemperatures of storage. After that the samples were separated usingSDS-PAGE and subsequently analyzed by Western blot. The EC50 wasdetermined and the values were compared in Table 1 (the average of 4experiments is given).

TABLE 1 EC50 EC50 Differentiation medium [pM] [mol/1] 1. Neurobasal +B-27 + GlutaMax + RA 0.22 2.18 × 10 -13 2. MEM + B-27 + NEAA + N-2 + RA1.51 1.51 × 10⁻¹² 3. MEM + B-27 + NEAA + N-2 + RA + B12 + 2.14 2.14 ×10⁻¹² Fe + Zn (low) 4. MEM + B-27 + NEAA + N-2 + RA + B12 + 1.51 1.51 ×10⁻¹² Fe + Zn (high) 5. MEM + B-27 + NEAA + N-2 + RA, diluted 0.24 2.37× 10 -13 to 225 mOsm/kg 6. Neurobasal + B-27 + GlutaMax + RA + Zn (high)0.27 2.65 × 10⁻¹³ Bold EC50 values = high sensitivity for BoNT

Surprisingly, the sensitivity of the cells to BoNT/A was increased by10-fold just by substituting MEM by Neurobasal medium (2. vs. 1.).Supplementing MEM with components of Neurobasal medium which MEM lacks(iron, vitamin B12 and zink) did not affect the susceptibility of thecells to BoNT/A (2. vs. 3. and 4.). Diluting MEM with sterile deionizedwater to 225 mOsm/kg, however, yielded the same surprising effect as theuse of Neurobasal medium. Thus, reducing the osmolality from 300 mOsm/kgto 225 mOsm/kg dramatically increased the sensitivity of SiMa cells from˜2 pM to ˜0.2 pM.

Table 2 shows the osmolality values of differentiation media 1 to 6.

TABLE 2 Osmolality of the tested differentiation media DifferentiationOsmolality medium Composition (mOsm/kg) 1 Neurobasal + B-27 + GlutaMax +RA 225 2 MEM + B-27 + NEAA + N-2 + RA 300 3 MEM + B-27 + NEAA + N-2 +RA + 300 B12 + Fe + Zn (low) 4 MEM + B-27 + NEAA + N-2 + RA + 300 B12 +Fe + Zn (high) 5 MEM + B-27 + NEAA + N-2 + RA, 225 diluted to 225mOsm/kg 6 Neurobasal + B-27 + GlutaMax + 225 RA + Zn (high)

For example, MEM has an osmolality of about 300 mOsm/kg. Usually, cellculture media used in the prior art have an osmolality between 290 and350 mOsm/kg. The osmolality of the cell culture media is not essentiallyinfluenced by other ingredients. For supplements, an osmolality withinthe desired range is chosen.

ii) P19 cells were cultivated according to the protocol of the DSMZ(German collection of Microorganisms and Cell cultures). 5 days beforeplating the cells on 96-well plates, the cells were transferred ontobacterial grade petri dishes and the medium was changed to 95% MEM-alpha(Gibco by Life Technologies™ #32571) supplemented with 5% FBS (PAA#A15-152) and 0.1 μM retinoic acid (RA; Sigma-Aldrich #R2625) as“priming medium”. During 5 days of growth the cells formed clumps oraggregates. Those were trypsinized and the cells (30.000 cells/cm²) wereplated on 96-well plates (TPP #92096) five days before addition ofBotulinum Neurotoxin (BoNT) in the above mentioned priming medium. 24 hafter plating the cells, the medium was exchanged for one of thefollowing differentiation media:

-   1. 97% Neurobasal (Gibco by Life Technologies™ #21103)+2% B-27    supplement (Gibco by Life Technologies™ #17504-044)+1% GlutaMAX™    (Gibco by Life Technologies™ #35050-038)+3 μM retinoic acid (RA;    Sigma-Aldrich #R2625); 225 mOsm/kg.-   2. 95% MEM-alpha (Gibco by Life Technologies™ #32571)+5% FBS (PAA    #A15-152)+0.1 μM retinoic acid (RA; Sigma-Aldrich #R2625); 300    mOsm/kg.-   3. 90% MEM-alpha (Gibco by Life Technologies™ #32571)+10% FBS (PAA    #A15-152); 300 mOsm/kg.-   4. 97% MEM-alpha (Gibco by Life Technologies™ #32571)+2% B-27    supplement (see above)+1% N-2 supplement (see above)+1% NEAA (see    above)+1 μM retinoic acid (RA; Sigma-Aldrich #R2625); 300 mOsm/kg.

Four days after the change to differentiation medium, half of the mediumper well is exchanged for fresh medium which contains BoNT/A indifferent concentrations (serial 1:2 dilution in 11 steps starting from10 pM plus negative control without BoNT/A). 72 h after addition ofBoNT, the medium was aspirated and lysis buffer containing benzonase wasadded. The plate was incubated at room temperature (RT) and thenRoti-load 1 (Roth #K929.1) was added. Samples prepared as aforementionedcould be stored at a temperature below −70° C. The low temperature iscritical because SNAP-25 seems to be specifically degraded at highertemperatures of storage. After that the samples were separated usingSDS-PAGE and subsequently analyzed by Western blot. The EC50 wasdetermined and the values were compared in Table 3 (the average of 4experiments is given).

TABLE 3 EC50 EC50 Differentiation medium [pM] [mol/1] 1. Neurobasal +B-27 + GlutaMax + RA 1.10 1.10 × 10 -12 2. α-MEM + 5% FBS + RA 8.84 8.84× 10⁻¹² 3. α-MEM + 10% FBS 17.7 1.77 × 10⁻¹¹ 4. α-MEM + B-27 + NEAA +N-2 + RA 12.5 1.25 × 10⁻¹¹ Bold EC50 values = high sensitivity for BoNT

The invention claimed is:
 1. A method for the generation ofneurotoxin-sensitive, neuronal differentiated cells comprising the stepsof: a) cultivating SiMa tumor cells in a culture medium under conditionsand for a period of time which primes the SiMa tumor cells for neuronaldifferentiation, wherein the culture medium comprises 80 to 98.8%OptiMEM, 1 to 10% Fetal Bovine Serum (FBS), 0.2 to 5% B27 supplementand/or 0.2 to 5% N2 supplement and, optionally, non-essential aminoacids and/or an antibiotic; b) adjusting the osmolality of adifferentiation medium comprising (i) B27 supplement and/or (ii) N2supplement to an osmolality of 180 to 230 mOsm/kg, wherein thedifferentiation medium comprises 78 to 98.3% neurobasal medium, 1 to 10%FBS, 0.5 to 2% GlutaMAX, 0.2 to 5% B27 supplement, and/or 0.2 to 5% N2supplement; and c) cultivating the SiMa tumor cells primed for neuronaldifferentiation in step a) for at least 3 days in the differentiationmedium of step b) having an osmolality of 180 to 230 mOsm/kg, wherebythe sensitivity to Clostridium neurotoxin in the neuronal differentiatedcells cultivated according to steps a) and c) is increased compared toSiMa tumor cells cultivated in a differentiation medium having anosmolality of about 290-350 mOsm/kg.
 2. The method of claim 1, whereinthe differentiation medium further comprises retinoic acid.
 3. Themethod of claim 2, wherein the retinoic acid is present in aconcentration of between 0.01 μM and 300 μM.
 4. The method of claim 1,wherein the differentiation medium further comprises an antibiotic agentand/or a cytostatic agent which inhibits growth of non-neuronal cells.5. The method of claim 1, wherein the differentiation medium furthercomprises ganglioside GT1b (GT1b).
 6. The method of claim 5, wherein theGT1b is present in a concentration of between 25 μM and 75 μM or ispresent in a concentration of 50 μM.
 7. The method of claim 1, whereinstep a) comprises reduction of serum from the culture medium and/oraddition of retinoic acid.
 8. The method of claim 1, wherein the tumorcells are SiMa cells (DSMZ ACC deposit number: 164).
 9. The method ofclaim 8, wherein the SiMa cells are cultivated in step a) for a periodof at least 36 hours.
 10. The method of claim 8, wherein the culturemedium in step a) further comprises an antibiotic agent and/ornon-essential amino acids (NEAA).
 11. The method of claim 8, wherein theSiMa cells are cultivated in step a) on tissue culture dishes which arecoated with at least one compound selected from the group consisting ofpoly-L-lysine, poly-D-lysine, collagen, laminins, and gelatin.
 12. Themethod of claim 1, wherein the osmolality of the differentiation mediumis about 225 mOsm/kg.